A second essential function of the Est1-binding arm of yeast telomerase RNA

Abstract

The enzymatic ribonucleoprotein telomerase maintains telomeres in many eukaryotes, including humans, and plays a central role in aging and cancer. Saccharomyces cerevisiae telomerase RNA, TLC1, is a flexible scaffold that tethers telomerase holoenzyme protein subunits to the complex. Here we test the hypothesis that a lengthy conserved region of the Est1-binding TLC1 arm contributes more than simply Est1-binding function. We separated Est1 binding from potential other functions by tethering TLC1 to Est1 via a heterologous RNA-protein binding module. We find that Est1-tethering rescues in vivo function of telomerase RNA alleles missing nucleotides specifically required for Est1 binding, but not those missing the entire conserved region. Notably, however, telomerase function is restored for this condition by expressing the arm of TLC1 in trans. Mutational analysis shows that the Second Essential Est1-arm Domain (SEED) maps to an internal loop of the arm, which SHAPE chemical mapping and 3D modeling suggest could be regulated by conformational change. Finally, we find that the SEED has an essential, Est1-independent role in telomerase function after telomerase recruitment to the telomere. The SEED may be required for establishing telomere extendibility or promoting telomerase RNP holoenzyme activity.

Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP

“…It is quite remarkable that this module, complete with the associated proteins Pop6, Pop7, and Pop1, has found its way into the telomerase RNP, a totally unrelated and structurally distinct entity but in which the P3 domain plays a similar role. Curiously, the telomerase RNP permits that the stem IVc arm that comprises the P3 domain and the Est1 binding site be relocated to other locations on the RNA and it can even be supplied in trans (Zappulla and Cech, 2004; Lebo et al, 2015). This permissiveness to a permutation of certain parts of the RNP may be a consequence of the proposed modular assembly.…”

Repositioning the Sm-binding site inS. cerevisiaetelomerase RNA reveals RNP organizational flexibility and Sm-directed 3′-end formation

“…Between the results presented here and previous repositioning studies for the binding sites of Est1 and Ku subunits (Zappulla and Cech 2004;Zappulla et al 2011), it is now evident that TLC1 is an organizationally flexible scaffold for these three well-established RNA-bound holoenzyme subunits of telomerase. While organizational flexibility has not yet been tested for the Pop1/Pop6/Pop7 complex that was recently reported to bind to an essential sequence in TLC1 near the Est1-binding site (Lemieux et al 2016), the entire Est1-Pop1/6/7-binding region of TLC1 has been shown to function when it is expressed in trans as a separate RNA while Est1 is artificially tethered near the 3¢ end of TLC1 (Lebo et al 2015), suggesting that the flexible scaffold model applies to the Pop1/6/7 proteins as well.…”

“…Northern blotting was performed as described previously (Zappulla et al 2005;Zappulla et al 2011;Hass and Zappulla 2015;Lebo et al 2015). Briefly, cells from the serial passaging plates were grown in liquid cultures to an OD600 of ~1.0 and harvested.…”

“…Southern blotting was performed as described previously (Zappulla et al 2005;Zappulla et al 2011;Hass and Zappulla 2015;Lebo et al 2015). Briefly, cells were grown and harvested in the same manner as those used for northern blots, and genomic DNA was isolated from these pellets (Gentra Puregene system).…”

Long non-coding RNAs as novel targets for therapy in hepatocellular carcinoma

“…ASO may be more effective than siRNAs for lncRNA targets in which the secondary structure of the lncRNA might preclude or limit optimal association with a siRNA. However, as with other ASO-targeting strategies, the adequacy of cellular uptake and potential for off-target effects needs to be considered (Gutschner et al, 2013; Kotake et al, 2011; Lebo, Niederer, & Zappulla, 2015; Zappulla & Cech, 2006). …”